Apparatuses, systems, and methods for culturing cells

ABSTRACT

Apparatuses, systems, and methods are provided for culturing a buoyant target tissue. Embodiments include a first surface configured to culture a first layer of supporting cells, and a second surface configured to culture a second layer of supporting cells. The first layer of supporting cells may be formed on a portion of the first surface and the second layer of supporting cells may be formed on a portion of the second surface. The buoyant target tissue may be added to the first layer of supporting cells. The second layer of supporting cells may be placed on the first layer of supporting cells such that the buoyant target tissue is sandwiched between the first layer of supporting cells and second layer of supporting cells.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/240,612, filed Oct. 13, 2015, which is incorporatedherein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows microscopic images of human white adipose tissue (WAT)cultured in a matrix of collagen after 2 days of conventional,collagen-embedded culture.

FIG. 1B shows microscopic images illustrating differences in morphologybetween differentiated “adipocytes” (diffAds) and primary adipocytes.

FIG. 2 is a graphical illustration comparing gene expression ofadipocyte identity genes between conventional culture models and primaryWAT.

FIG. 3 is an isometric view of a culture apparatus having a layer ofsupporting cells disposed in a thermoresponsive dish and an insertdevice for removing the layer of supporting cells from the dish,according to an exemplary embodiment of the present disclosure.

FIG. 4 is an isometric view of the insert device shown in FIG. 3 withthe layer of supporting cells attached to a base of the insert device.

FIG. 5 is an isometric view of the culture apparatus shown in FIG. 4,with the layer of supporting cells attached to the base of the insertdevice being removed from the thermoresponsive dish.

FIG. 6 is an isometric view of a culture apparatus having a buoyant cellor tissue explant deposited on a first layer of supporting cells in athermoresponsive dish, according to an exemplary embodiment of thepresent disclosure.

FIG. 7 is an isometric view of the culture apparatus shown in FIG. 6 andan insert device with a second layer of supporting cells attached to abase of the insert device to be deposited on top of the tissue explantand first layer of supporting cells, according to an exemplaryembodiment of the present disclosure.

FIG. 8 is an isometric view of white adipose tissue sandwiched betweentwo layers of supporting cells, according to an exemplary embodiment ofthe present disclosure.

FIG. 9 shows microscopic images illustrating the morphologic stabilityof a sandwiched White Adipose Tissue (SWAT) cell culture system,according to an exemplary embodiment of the present disclosure.

FIG. 9A shows microscopic images of a SWAT culture system having abi-layer of supporting cells surrounding a WAT cell cluster, accordingto an exemplary embodiment of the present disclosure.

FIG. 9B shows microscopic images illustrating the morphologic stabilityof WAT cell clusters in the SWAT cell culture system, according to anexemplary embodiment of the present disclosure.

FIG. 10 is a series of microscopic images showing the long-termstability of a WAT cell cluster in a SWAT culture system, according toan exemplary embodiment of the present disclosure.

FIG. 11 is a graphical illustration showing that SWAT cultures aretranscriptionally active and express genes associated with adiposetissue identity, according to an exemplary embodiment of the presentdisclosure.

FIG. 12 shows microscopic images indicating that SWAT cultures aretranslationally active and express protein associated with adiposetissue identity, according to an exemplary embodiment of the presentdisclosure.

FIG. 13A is a graphical illustration showing that SWAT cultures secreteleptin at basal levels on days 1 and 5 of culture, which mirrors primaryWAT, according to an exemplary embodiment of the present disclosure.

FIG. 13B is a graphical illustration showing that SWAT cultures secreteadiponectin at basal levels on days 1 and 5 of culture, which mirrorsprimary WAT, according to an exemplary embodiment of the presentdisclosure.

FIG. 13C is a graphical illustration showing that SWAT cultures performlipolysis in response to catecholamine stimulation after days 1 and 5 inculture, according to an exemplary embodiment of the present disclosure.

FIG. 14 shows images illustrating that SWAT cultures fully engraft intoimmunocompromised, eGFP-labeled mice, according to an exemplaryembodiment of the present disclosure.

DESCRIPTION OF EMBODIMENTS

Obesity is an increasingly common condition afflicting over 79 millionAmericans. Obesity may be associated with various diseases including:type 2 diabetes, heart disease, stroke, arthritis, and some cancers. Inaddition to the health impact, the direct financial cost related totreatment of obesity and related diseases is estimated to exceed $150billion in the United States alone. Presently, there is a strong needfor anti-obesity therapeutics approved for human intervention.

Obesity may be described as an overgrowth of white adipose tissue (WAT)in the body. In general, WAT may be considered an organ in the humanbody, functioning as an energy reservoir where extra calories may bestored. WAT is found throughout the human body and may be subcutaneousin origin, or originate from a variety of anatomical areas including,intra alia, the abdomen, chest, gluteus, and limbs. WAT may also beconsidered an endocrine organ that produces hormones to regulatemultiple physiological systems, e.g., hunger/satiety, glucosemetabolism, and lipid metabolism. A properly functioning WAT organ iscritical. Indeed, insufficient WAT may lead to illness or death.

As an organ, WAT includes mature, adipocytes (wAds) that may bedescribed morphologically as large cells having a unilocular lipiddroplet that exceeds 95% of cellular volume. The presence of this largelipid droplet renders wAds buoyant. Human wAds may also be considered asexceptionally fragile cells due in large part to their size. Forexample, human wAds range in size from about 100 to about 140 μm insize, which is nine (9) times the volume of rodent wAds.

Attempts to culture primary, human wAds have largely been unsuccessful.Conventional in vitro culture methodologies employ techniques such asenzymatic treatment and mechanical handling to dissociate primary WATtissue and isolate wAds. This treatment typically destroys or severelydamages a large proportion of the wAds, wherein the majority of wAdsundergo cell lysis within 72 hours after handling. Accordingly, researchmodels of human WAT derived from wAds do not exist.

Attempts to overcome the challenge associated with wAds culture includeembedding wAds in a matrix of collagen protein. However, this techniquehas had limited success. FIG. 1 shows micrographs of collagen-embeddedhuman WAT stained with propidium iodine indicating the induction ofprogrammed cellular death, a.k.a., apoptosis, after 2 days of culture.In addition to their fragility, adipocytes are also consideredterminally differentiated and mitotically inactive. Therefore, wAds maynot be expanded in culture without altering their differentiation state,i.e., dedifferentiating.

Unlike most other model cell types, for which stocks of cells may befrozen for long-term storage, human WAT/wAds must be obtained fresh fromthe operating room or clinic and used immediately for each experiment.Researchers must, therefore, rely on surgically procured, human WATtissue as a source material, which limits the availability of WAT/wAdsto non-clinician researchers. In fact, researchers lacking relationshipswith clinicians may not have access to human WAT. Research experimentsmay then become tied to clinician schedules, which can be unpredictable.Further, tissue procurement may be time-consuming, and often requirestravel, donning of surgical attire, and hospital approval ofinvestigational protocols. These barriers to accessing source WAT haveslowed the overall pace of scientific discovery and may deterresearchers from investigating the biology of human WAT altogether.

Currently, researchers rely on models including rodent models orstromal/stem cell models chemically differentiated into adipocytes(i.e., diffAds). However, these experimental models fail to recapitulateprimary, human WAT biology. For example, one of the first-identifiedanti-obesity pathways was controlled by beta-3 adrenoreceptors (β3-ARs).Using selective β3-AR agonists, obesity and diabetes were successfullycured in several rodent models. However, the same selective β3-AR thatwere successful in rodent models of obesity had little activity againsthuman β3-ARs, resulting in multiple failed clinical trials.

Similarly, certain model cell types, e.g., stromal and stem cells, maybe chemically differentiated into adipocytes (diffAds). However, diffAdsonly express human, wAds markers, including CCAAT/enhancer-bindingprotein alpha, lipoprotein lipase, fatty acid binding protein 4, andhormone sensitive lipase, at a reduced levels. Further, models ofobesity based on diffAds fail to recapitulate wAds functionality inmetabolic assays measuring glycerol release, adiponectin release, andglucose uptake.

Genetically, in vitro cell models do not share similar gene expressionpatterns with wAds. FIG. 2 graphically illustrates the variation inadipocyte identity gene expression between conventional culture modelsand primary WAT. The Green bars within FIG. 2 represent the expressionlevels of stem cells differentiated into multiloculated “adipocytes”(diffAds) using standard protocols. The Red bars within FIG. 2 representthe expression levels of stem cells differentiated into “adipocytes”using PPARg-expressing lentivirus constructs. The Blue bars within FIG.2 represent the expression levels of primary adipose tissue. As shown,primary adipose tissue expresses adipocyte genes at levels 10 to 100times greater than in vitro models. Accordingly, conventional modelsfail to recapitulate the gene expression levels of primary adiposetissues.

Apparatuses, methods and systems are provided for culturing tissues andcells. In exemplary, non-limiting embodiments, apparatuses, methods andsystems are provided for culturing buoyant, primary, human tissueexplants and cells under conditions capable of maintaining primary celltype characteristics, including morphology, gene and protein expressionlevels, and metabolic function, even after extended periods of time inculture.

In exemplary embodiments, apparatuses, methods, and systems for in vitroculture of buoyant tissue explants of human WAT are provided.

Embodiments of the present disclosure provide systems, methods, andapparatuses for culturing buoyant tissues and cells when added to anaqueous culture medium. Embodiments of the present disclosure providesystems, methods, and apparatuses for culturing primary, human tissueexplants and cells obtained from individuals, e.g., patients.Embodiments of the present disclosure provide systems, methods, andapparatuses for culturing human tissue and cells for extended periods oftime, e.g., several weeks, in a stable, undifferentiated state.

Embodiments of the present disclosure provide systems, methods, andapparatuses for configuring a micro-physiological, e.g.,organ-on-a-chip, model system. Embodiments of the present disclosureprovide for the evaluation of the effect of chemical compounds, such aspharmaceuticals, on human tissue explants and cells cultured via thesystems, methods, and apparatuses disclosed herein.

Generally, in vitro tissue and cell culture systems employ culturevessels, e.g., dishes, plates, flasks, slides, to which tissues or cellsare added along with a nutrient rich medium. In certain instances,culture dishes may provide a substrate to which tissues or cells mayadhere, and the medium may provide the necessary components to supportand promote metabolic function of the tissues and cells added thereto.Establishing a new culture of tissue or cells requires transferringsample tissues or cells to a culture dish having an aqueous culturemedium. Tissue and cell types that are not buoyant may come to rest onthe surface of a culture vessel where tissue or cellular attachment tothe surface occurs through a complex process commonly referred to ascellular adhesion. However, certain tissue and cell types are buoyant,and therefore, float in their culture medium rather than adhere to thesurface of a culture dish. For certain cell types, failure to adhere mayresult in cell death.

Embodiments of the present disclosure provide systems, methods, andapparatuses for in vitro culture of buoyant tissues and cells. Inparticular embodiments, the systems, methods, and apparatuses describedherein may be adapted to culture all tissue and cell types including butnot limited to: white adipose tissue (WAT), brown adipose tissue (BAT),brain, nervous system tissue, thyroid, pancreas, spleen, cartilage,liver, kidney, and bone.

Referring to FIGS. 3 to 8, different views of a system for culturingbuoyant cell types is shown. FIG. 3 illustrates an exemplary embodimentof a culture apparatus 500 for culturing buoyant cell types. Cultureapparatus 500 may include a culture vessel 200, e.g., a culture dish200, and an insert 100. In certain non-limiting embodiments, the culturevessel 200 and insert 100 may comprise materials that promote adhesionof tissues or cells to the surface of the vessel or the insert.

In some embodiments, adhesion-promoting materials may be a component ofthe vessel 200 or insert 100, per se. In other embodiments,adhesion-promoting materials may be added to the vessel 200 and/orinsert 100. In this embodiment, the respective base/surface of thevessel 200 and/or insert 100 may be coated with a matrix of proteins orextracellular material. Non-limiting examples of adhesion promotingmaterials include but are not limited to poly(N-isopropylacrylamide)(pNIPAAM/pNIPAm), modified methylcellulose, and thermoresponsivematerials, e.g., thermoresponsive polyelectrolyte multilayer films,gelatin, collagen, hyaluronic acid, and cellulose.

In an exemplary embodiment of the present disclosure, a culture vessel200 may include a culture dish 200 having a base 201, side walls 203,and an opening 202. Base 201 of culture dish 200 may be configured toallow culture of at least one layer of supporting cells 204. Culturedish 200 may be configured to include an opening 202 for insertion of aninsert 100 device.

As shown in FIG. 3, insert 100 may include a base 102 and a handle 101.Handle 101 may be configured to allow insertion through the culture dishopening 202. Base 102 may be configured to allow culture of at least onelayer of supporting cells 104.

In various embodiments, a thermoresponsive layer may be added to thesurface of a culture dish base 201 prior to culturing supporting cells.A layer of supporting cells 204 may be cultured on the surface of thethermoresponsive layer covering a culture dish base 201 or an insertsurface 102. See, FIG. 3. Treatment of the culture dish 200 and theinsert 100 surfaces with a thermoresponsive layer may allow transfer ofan intact layer of supporting cells from either a culture dish 200 orthe insert 100 to another culture dish or insert. See, FIG. 3, FIG. 4and FIG. 5.

In an exemplary embodiment, a first culture dish 200 having a layer ofthermoresponsive material on the surface of a first culture dish base201 may be used to culture a layer of supporting cells 204. Insert 100may be placed through the opening 202 of the first culture dish 200 suchthat the surface 102 of the insert 100 may contact the layer ofsupporting cells 204. Conditions in the culture environment may bealtered to activate the thermoresponsive material, e.g., change intemperature, and release the supporting cell layer 204 from the firstculture dish 200 base surface 201, allowing adherence of the supportingcell layer 204 to the surface 102 of the insert 100. See, FIG. 4.Supporting cells 204 having been removed from a culture dish 200 basesurface 201 by activation of a thermoresponsive material, and attachedto the surface 102 of the insert 100 are also shown in FIG. 5.

In an exemplary embodiment, subcutaneous WAT samples may be procuredfrom human subjects during elective surgical procedures. In thisembodiment, sample sizes may range from about 100 to about 5000 grams ofWAT tissue. In a particular embodiment, an experimental sample may bedivided for various experimental purposes. In a particular embodiment, aportion of the subcutaneous WAT sample, e.g., 10 grams, may be minced,flash frozen, and stored as a matched primary WAT sample. In aparticular embodiment, a portion of the subcutaneous WAT sample, e.g.,10 grams, may be stored in a nucleic acid lysis buffer, e.g., RNeasyLipid Tissue Mini Kit™ (Qiagen), as a matched primary WAT sample fortranscriptional confirmation. In particular embodiment, a portion of thesubcutaneous WAT sample, e.g., 25 grams, may be used to produce SWATcultures according to embodiments of the present disclosure. In aparticular embodiment, a portion of the subcutaneous WAT sample, e.g.,25 grams, may be used to isolate matched supporting cells, e.g.,adipocites ADSCs for differentiation into diffAds using a standardprotocol when the WAT is minced, enzymatically digested, andcentrifuged.

In an exemplary embodiment of the present disclosure, primary, human WATmay be isolated from a patient and mechanically minced into segments300. WAT tissue segments 300 may be transferred to a culture dish 400having a layer of supporting cells 304 growing on a culture dish base401. See, FIG. 6.

Insert 100 having a layer of supporting cells 204 may then be insertedinto culture dish 400 including WAT tissue 300 atop a layer ofsupporting cells 204. See, FIG. 7. In this embodiment, the primary,human WAT tissue 300 may then be sandwiched between two layers ofsupporting cells, 204, 304, to form a sandwich WAT (SWAT) co-culturesystem 800. See, FIG. 8. Upper supporting cell layer 104 may be attachedto the base 102 of insert 100 and serve to hold the buoyant WAT tissue300 in contact with the underlying layer of supporting cells 204attached to culture dish 200 until adhesion occurs. In an exemplaryembodiment, adhesion between the WAT tissue 300 and the layers ofsupporting cells 104, 204 occurs within minutes. In certain embodiments,the bi-layer construct of the SWAT system may be entirely cellular ormay contain various synthetic or acellular components.

In an exemplary embodiment, 0.5-1 mm segments of human, primary WATtissue are sandwiched between two layers of supporting cells, e.g.,adipose-derived stromal cells (ADSCs), to form the SWAT co-culturesystem described herein. Supporting cells, e.g., ADSCs, may be culturedon standard tissue culture plates coated with a thermoresponsivesubstrate. The SWAT culture system as disclosed herein may also astandard culture media. Examples of standard culture media including atleast low glucose DMEM, about 10% newborn calf serum, and about 1%penicillin/streptomycin antibiotic solution.

In exemplary embodiments, the SWAT system described herein may beutilized as a test model for any extrinsic factor or system intended tomodify the biology or physiology of adipose tissue or adipocytes. Invarious embodiments, test factors may be introduced to the cell culturemedium and their impact evaluated in the isolated human adipocytes orsegments of primary, human WAT. Non-limiting embodiments of test factorsmay include but are not limited to pharmaceutical compounds, recombinantor native viruses, recombinant or isolated nucleic acid constructs,expression vectors, siRNA construction, micro RNA constructs, genetictools, bacteria, and environmental modulations including temperature,pressure, and modulation of gases.

As illustrated in the micrographs of FIG. 9A, a SWAT culture isestablished between a bi-layer of supporting cells. The WAT is added toa bottom layer of unlabeled supporting cells and a top layer ofsupporting cells expressing enhanced green fluorescent protein (eGFP) isadded. As shown in FIG. 9A, a WAT cluster of cells is sandwiched betweena bi-layer of supporting cells, e.g., eGFP negative (bottom layer) andeGFP positive (top layer) to forming an exemplary SWAT culture system.Moreover, FIG. 9B shows microscopic images of a SWAT culture over time.The WAT cell clusters within SWAT culture are capable of retaining theirmorphologic stability for up to at least 47 days or about 6.7 weeks.See, FIG. 9B.

In various embodiments, the SWAT co-culture system described hereindemonstrate long-term viability and stability which are importantfeatures for micro-physiologic models of terminally differentiated cellsincluding WAT. In an exemplary embodiment of the present disclosure,long-term morphological stability is illustrated in staining of SWATclusters in FIG. 10. Structural stability of the WAT cell clusters isdemonstrated in FIG. 10A by restriction of neutral lipids to only WATcells even after 51-days in SWAT culture. Likewise, propinium iodinestaining of WAT cells in a SWAT culture was negative as seen in FIG.10B. Propinium iodine negative WAT cells indicates that, after at least18 days of SWAT culture, WAT cells were not undergoing programmed celldeath, i.e., apoptosis. Finally, restriction of lipophilic staining tothe adipocytes of the SWAT co-cultures further evidences the long-termviability of the systems and methods described herein. See, FIG. 10C. Incontrast to conventional methods, an exemplary embodiment of the presentdisclosure demonstrates that the WAT cell clusters within SWAT culturesmaintain their intracellular architecture, e.g., FIG. 10A, are viableand not entering a state of programmed cell death, e.g., FIG. 10B, andare maintained as separate populations.

FIG. 11 demonstrates that the SWAT system described herein is capable ofmaintaining a gene expression profile of at least six (6) key adipocyteidentity genes including: activated receptor gamma (PPARy), which is amaster regulator of adipocyte differentiation/identity; fatty acidbinding protein 4 (FABP4), which is a transcription factor necessary forterminal adipocyte differentiation; CCAAT/enhancer-binding protein alpha(CEBPα), which delivers long-chain fatty acids and retinoic acid tonuclear receptors; lipoprotein lipase (LPL), which is an enzyme thathydrolyses triglycerides; hormone sensitive lipase (HSL), whichhydrolyzes stored triglycerides to free fatty acids; and adiponectin(ADIPOQ), which is a central adipokine in the control of fat metabolismand insulin sensitivity. Experimentally, total RNA was collected fromSWAT cultures and expression levels were compared to subject-matchedprimary WAT using reverse transcription polymerase chain reaction(RT-PCR). At a transcriptional level, the SWAT culture system of thepresent disclosure maintains the adipose tissue identity. See, FIG. 11.

At a translational level, the SWAT culture system of the presentdisclosure also maintains adipocyte proteins. As seen in FIG. 12,immunocytochemistry staining of SWAT cultures demonstrates the proteinproduction of adipocyte markers including: PPARg, FABP4, beta-3adrenergic receptor (B3-AR), which is associated with lipolysis inadipocytes, and perillipin, which is also known as protein lipiddroplet-associated protein and coats lipid droplets in adipocytes.

In addition to expressing gene and protein markers associated withadipocytes, SWAT cultures of the present disclosure also perform basalendocrine functions. In certain embodiments of the present disclosure,it may be desirable to maintain the functionality of tissues and cellsin culture models as close the native tissue as possible. In variousembodiments, SWAT clusters maintain their native endocrine function.Primary, human WAT is an endocrine tissue which secretes at least 2hormones including: leptin and adiponectin. Based on normalized,quantitative ELISA assays, as illustrated in FIG. 13A and FIG. 13B, SWATcultures secrete leptin and adiponectin at similar levels assubject-matched WAT after both one (1) and five (5) days in culture.See, FIG. 13A and FIG. 13B.

Further, the SWAT culture system described herein performed lipolysis atlevels similar to primary WAT in response to exogenous signals. See,FIG. 13C. Lipolysis, which is the process of converting stored fats intometabolic fuel, is a central function of WAT. In vivo, lipolysis occursat a basal rate and is upregulated by catecholamines. In vitro,lipolysis can be quantified by measuring the amount of free glycerolreleased with normalization to total protein levels using a conventionalBradford total protein assay. In a particular embodiment of the presentdisclosure, SWAT cultures were exposed to 100 μM forskolin+1 μMepinephrine for three (3) hours. After 1-day and 5-days in culture, SWATcultures performed lipolysis at levels similar to primary WAT inresponse to stimulation.

In another exemplary embodiment, the SWAT culture system describedherein may maintain native functionality after at least ten days of SWATculture. As shown in FIG. 14, a SWAT culture was harvested after tendays of SWAT culture and subcutaneously injected into immunocompromisedeGFP-labeled mice (NOD-scid IL2Rγnull). It is known that implantedtissue must recruit a new blood supply, i.e., induce vascularization, orthe tissue will die, i.e., necrosis, within 48 hours and subsequently beliquefied by the host. In an exemplary embodiment, SWAT transplants werere-harvested from their mice hosts ten days after subcutaneousinjection. Upon visual examination, the injected SWAT tissues werereadily seen by the naked eye. See, FIG. 14 (Re-harvest). Further,histological analysis revealed that the SWAT transplants retained thearchitecture characteristic of WAT, and did not express eGFP endogenousto the mouse host. See, FIG. 14 (SWAT and Neg). This data indicates thatSWAT cultures may retain sufficient native functionality to enable SWATtissue engraftment even after ten days in SWAT culture. Becauserecruitment of new, host blood supply, i.e. vascularization, is a highlycomplex process, this data further indicates that the SWAT systemdescribed herein may be a robust, micro-physiologic model of human WAT.

Exemplary embodiments of the present disclosure provide a system thatmay allow investigation into effective anti-obesity strategies. It waspreviously understood that only brown adipose tissue (BAT) was capableof burning energy in a process known as thermogenesis. However, in bothrodents and diffAds models, it is known that white adipocytes (wAds)could be induced to become thermogenic beige/“brite” adipocytes (brAds),which may be identified biochemically based on an upregulation ofuncoupling protein 1 (UCP1) in response to elevated intracellular cyclicAMP (cAMP) levels. Specifically, induction of UCP1 transforms WAT intothermogenic cells and leads to an alternation in the cellularmorphology.

Morphologically, brAds shift from a large, unilocular phenotypeassociated with WAT cells to a multilocular phenotype. The WAT-specificsource of brAds has been confirmed by lineage tracing studies inrodents: brAds are myogenic factor 5 (Myf5) negative whereas brownadipocytes (bAds) share a Myf5+ lineage with skeletal myocytes. Inrodents, browning has been observed in most subcutaneous and visceralWAT depots. In rodent models, the weight-loss incurred by browned WATcan be profound. Accordingly, the SWAT culture system as disclosedherein may provide a micro-physiological model system for evaluatingcontrolled browning of culturing primary, human WAT as a feasible andeffective anti-obesity strategy.

Embodiments of the present disclosure provide systems and methods forinvestigating the biochemistry of browning pathways identified in rodentand diffAds models which may be controlled by: beta-3 adrenoreceptors(β3-ARs), cold receptors, cardiac natriuretic receptors, Janus inhibitorkinase 3 (JAK3), and Notch 1. Each of these endogenous biochemicalpathways provide numerous candidate targets for pharmaceuticalintervention. Several compounds have browned WAT in rodents and indiffAds. In rodents, chemical induction of browned WAT successfullyameliorated obesity and cured type 2 diabetes. Accordingly, the SWATculture system as disclosed herein may provide a micro-physiologicalmodel system for evaluating candidate pharmaceuticals in primary, humanWAT tissues.

Exemplary embodiments of the present disclosure provide an in vitrosystem that may allow investigation and evaluation into the effects ofchemical compounds, such as pharmaceuticals, on human WAT and otherbuoyant cell types. Non-limiting, exemplary candidate pharmaceuticalsmay include but are not limited to: agonists and antagonists of beta-3adrenoreceptors, e.g., 1&3-ARs; migrabegron, which is a 4th generation1&3 agonist FDA-approved for use in overactive bladder syndrome, but isknown to activates BAT in humans; CL-316243, which is a specific 1&3agonist, e.g., 1&1, 1&2, 1&3=0:1:100,000) that ameliorated obesity inobese, diabetic yellow KK mice; L-796568, which is abenzenesulfonamide-family specific 1&3 agonist, e.g., 1&1, 1&2,1&3=1:230:660, that improved energy expenditure but did not generatenotable anti-obesity effects in obese human males; BRL 26830A, which isa 1&3 agonist that demonstrated significant improvements in weight lossin a double-blinded trial.

In other exemplary embodiments, the culture system described herein maymaintain native functionality of other buoyant cells in culture.Endogenous biochemical pathways may be evaluated for pharmaceuticalintervention by applying and evaluating the impact of exogenous stimuli,e.g., chemical compounds. Any buoyant cell type, regardless of tissuetype or species of origin, may be a candidate for use in embodiments ofthe present disclosure. Exemplary embodiments of buoyant tissues andcell types that may be candidates for evaluation using the apparatuses,systems and methods disclosed herein include but are not limited to:hepatocytes, renal tissue and cells, brain tissue and cells, thyroidtissue and cells, splenic tissue and cells, liver tissue and cells,central and peripheral nervous tissue and cells, and immunologic tissueand cells. Moreover, buoyant cells may be obtained from any sourceorganism. Exemplary source organisms may include but are not limited to:plants, animals, protists, fungi, archaebacteria, and eubacteria.Additional exemplary sources of tissue or cells for evaluation using theapparatuses, systems and methods disclosed herein include but are notlimited to: human, mouse, rat, monkey, dog, cat, pig, non-humanprimates, and fish.

Exemplary embodiments of the present disclosure provide systems andmethods for investigating the biological responses of exemplary,non-limiting cell types. For example, an established buoyant tissue typemay include neuronal tissue. Neuronal tissue may not readily adhere tothe surface of culture dishes if, for example, excessive bubbles areintroduced to an aqueous culture media. Accordingly, the buoyant tissueculture apparatuses, systems and methods disclosed herein may bedirectly applied to the study of neuronal tissue.

Embodiments of the present disclosure provide apparatuses, systems andmethods for culturing neuronal tissue which may include embryonic oradult neuronal tissues. In an exemplary embodiment, the presentdisclosure provides a model system which may be used in the evaluationof neurogenesis. In other embodiments, the present disclosure mayprovide a system in which neuronal disease progress may be evaluated.

In an exemplary embodiment, the apparatuses, systems and methods of thepresent disclosure may be used to evaluate the biochemical pathwaysleading to the neuronal disease commonly known as Alzheimer's Disease(AD) and as well as the impact of various pharmaceutical interventions.For example, central to AD disease is the differential processing of theintegral membrane protein Amyloid Precursor Protein (APP) in the normalversus disease state. In the normal state, APP is initially cleaved byα-secretase to generate sAPP and a C83 carboxy-terminal fragment. Thepresence of sAPP is associated with normal synaptic signaling andresults in synaptic plasticity, learning and memory, emotionalbehaviors, and neuronal survival. In the disease state, APP is cleavedsequentially by α-secretase and γ-secretase to release an extracellularfragment called A 40/42. This neurotoxic fragment frequently aggregatesand results in A 40/42 oligomerization and plaque formation. A 40/42aggregation results in blocked ion channels, disruption of calciumhomeostasis, mitochondrial oxidative stress, impaired energy metabolismand abnormal glucose regulation, and ultimately neuronal cell death. Themicro-physiological system of the present disclosure provides a modelfor quickly and efficiently assessing buoyant neuronal tissues in vitrowhile maintaining the neuronal tissue in a native state.

Embodiments of the present disclosure provide apparatuses, systems andmethods for evaluating the biochemical pathways involved incardiovascular disease (CVD). Cardiovascular disease (CVD) remains theleading cause of death in the United States, with over 600,000 deathsper year and annual direct costs near $300 billion. High blood pressure(HTN) and obesity are two of the most prevalent and modifiable riskfactors for CVD. HTN affects 29.1% of adult Americans and successfullytreating blood pressure decreases CVD risk by 20-50%. Obesity is moreprevalent than HTN, affects 36% of adult Americans and is considered aglobal epidemic. However, while several classes of anti-hypertensivemedications are available, no broadly effective anti-obesity medicationshave been approved for patient use.

In an exemplary embodiment, the apparatuses, systems and methods of thepresent disclosure may be used to evaluate the overlapping biochemicalpathways involved in cardiovascular disease and obesity. For example,the pathogenesis of HTN often involves over-activation of therenin-angiotensin system (RAS). RAS over-activation has also been linkedto obesity, a disease involving the overgrowth of WAT. Moreover, the RASshares biochemical signaling pathways which overlap with obesitybiochemical pathways as evidenced by the fact that: (i) the molecularcomponents of RAS are present in adipose tissue, (ii) WAT secretesangiotensinogen (AGT), (iii) angiotensin II (Ang II) may induceadipogenesis in isolated adipocytes and differentiated adipocytes(diffAds), (iv) Ang II stimulation inhibited lipolysis in ex vivo humanadipocytes, thus favoring adipogenesis.

Moreover, embodiments of the present disclosure confirmed that SWATcultures are capable of maintaining RAS pathway constituent expression.For example, using RT-PCR it was determined that SWAT cultures preservesexpression of key RAS components (n=5): (i) SWAT AGT expression: 62% ofprimary WAT (range 47-79%); SWAT ACE expression: 58% of primary WAT(range 45-71%), SWAT AT1R expression: 14% of primary; WAT (range 6-19%);SWAT AT2R expression: 231% of primary WAT (range 72-617%), SWAT Renin:no detectable expression. Further, in terms of endocrine function, SWATsecretes AGT, leptin and adiponectin as determined via enzyme-linkedimmunosorbent assays (ELISA). Finally, SWAT secretes 77 to 204 ng AGTper mg of total protein, and ELISA testing identified no Ang II in mediafrom cultured SWAT. Together, this data indicates that RASover-activation may drive adipogenesis in both systemic and autocrinefashion. In embodiments, RAS inhibition through current, approvedpharmacotherapies may ameliorate both hypertension and obesity.Embodiments of the present disclosure provide apparatuses, systems, andmethods for investigation into this system.

While the embodiments are described with reference to variousimplementations and exploitations, it will be understood that theseembodiments are illustrative and that the scope of the inventions is notlimited to them. Many variations, modifications, additions, andimprovements are possible. Further still, any steps described herein maybe carried out in any desired order, and any desired steps may be addedor deleted.

What is claimed is:
 1. A cell culture apparatus for culturing a buoyanttarget tissue, comprising: a first surface configured to culture a firstlayer of supporting cells; and a second surface configured to culture asecond layer of supporting cells, wherein the first layer of supportingcells is formed on a portion of the first surface and the second layerof supporting cells is formed on a portion of the second surface,wherein the buoyant target tissue is added to the first layer ofsupporting cells, and the second layer of supporting cells is placed onthe first layer of supporting cells such that the buoyant target tissueis sandwiched between the first layer of supporting cells and secondlayer of supporting cells.
 2. The apparatus of claim 1, wherein thetarget tissue includes a buoyant cell type.
 3. The apparatus of claim 2,wherein the target tissue is derived from at least one of a eukaryote, aprokaryote, a mammal, a human, a rodent, a yeast, a bacteria, a plant,and a fungi.
 4. The apparatus of claim 3, wherein the target tissue isderived from a human.
 5. The apparatus of claim 4, wherein the humantarget tissue includes white adipocytes, individually isolatedadipocytes, brown adipocytes, human hepatocytes, human renal cells,human brain cells, human thyroid cells, human splenic cells, andimmunologic cells.
 6. The apparatus of claim 1, wherein at least one ofthe first and second layers of supporting cells include adiposetissue-derived stem cells, muscle cells, pluripotent cells includingembryonic stem cells, multipotent cells including adipose derived stemcells, neuronal stem cells and muscle stem cells, stromal cellsincluding fibroblasts, pericytes, and 3T3-L1.
 7. The apparatus of claim1, further comprising a medium for culturing the target tissue and thefirst and second layer of supporting cells.
 8. The apparatus of claim 1,wherein at least one of the first surface and second surface includes athermo-responsive substrate.
 9. A method for culturing a buoyant targettissue, comprising: culturing a first and a second population ofsupporting cells; adding the buoyant target tissue to the firstpopulation of supporting cells; and adding the second population ofsupporting cells to the first population of supporting cells and buoyanttarget tissue, wherein the buoyant target tissue is sandwiched betweenthe first and the second population of supporting cells.
 10. The methodof claim 9, wherein the first population of supporting cells is culturedin a vessel including adhesion supporting materials
 11. The method ofclaim 10, wherein the adhesion supporting materials include at least oneof a poly(N-isopropylacrylamide), a modified methylcellulose, athermoresponsive substrate, and gelatin.
 12. The method of claim 9,wherein the target tissue includes a buoyant or a non-buoyant tissue.13. The method of claim 12, wherein the target tissue includes whiteadipocytes, individually isolated adipocytes, brown adipocytes, humanhepatocytes, human renal cells, human brain cells, human thyroid cells,human splenic cells, and immunologic cells.
 14. The method of claim 10,wherein at least one of the first and second populations of supportingcells include adipose tissue-derived stem cells, muscle cells,pluripotent cells including embryonic stem cells, multipotent cellsincluding adipose derived stem cells, neuronal stem cells and musclestem cells, stromal cells including fibroblasts, pericytes, and 3T3-L1.15. A micro-physiological system, comprising: a culture of primary,human white adipose tissue (WAT) sandwiched between two layers ofsupporting cells; an exogenous agent configured to be added to theculture of WAT; and at least one detector configure to evaluate aphysiological change as a result of adding the exogenous agent to theWAT.
 16. The system of claim 15, wherein the WAT culture is maintainedin a viable, undifferentiated state for at least three days.
 17. Thesystem of claim 15, wherein the exogenous agent includes at least one ofa chemical, a pharmaceutical, a nucleic acid, a protein, a virus, alipid and a vector.
 18. The system of claim 15, wherein the detectorincludes at least one of an optical detector, a biochemical detector,and an energy detector.
 19. The system of claim 15, whereinphysiological changes include an intracellular change, an extracellularchange, a transcriptional change, a translational change, apost-transcriptional change, a post-translational change, a secretionchange, and functional change.